Beta thalassemia is one of the most prevalent forms of heritable blood disorders in the world. It is caused by mutations in human beta globin genes that result in reduced or abolished beta globin synthesis. Without beta globin chains to pair with, excess alpha globin chains are susceptible to oxidation to hemichromes, precipitate and damage red blood cell precursors as well as mature red blood cells, leading to ineffective erythropoiesis and profound anemia. Patients afflicted with the most severe forms of beta thalassemia require lifelong blood transfusions and iron chelation treatment. The only cure at present is BM transplantation with histocompatible donor cells, a limited option for many adult patients. The effort proposed here aim to ultimately, significantly improve the clinical picture in all patients with no transplant option by identifying an optimal targeted genetic engineering approach to do so. We will explore, side-by-side, three promising approaches alone and/or in certain combinations: 1) to reactivate developmentally silent gamma globin to pair with excess alpha globin (SA#1); 2) to downregulate alpha globin synthesis (SA#2); and 3) to drive targeted insertion of a therapeutic gamma globin gene into the beta globin locus (SA#3). For this purpose we plan to utilize an open-source targeted genome engineering platform, TALE effector nucleases (TALENs) to edit the relevant genomic loci of primary normal or patient mobilized peripheral blood (MPB) hCD34+ cells. Our intended genetic targets include a) potent gamma globin repressors BCL11A and its enhancer, as well as KLF1 (3 target sites), b) putative gamma globin repressor binding sites within the beta globin locus (3 target sites), c) the binding sites f alpha globin transactivator KLF4 in alpha globin promoters (3 target sites), d) alpha+ thalassemia- associated genomic sites in alpha globin locus (4 target sites), and d) two putative sites within beta globin locus for the insertion of a therapeutic gamma globin gene (2 target sites). The efficiency of editing and general effects on globin expression, erythropoiesis, and other potential side-effects will first be evaluated using normal MPB hCD34+ cells. The durability of editing and globin modulation will be studied by transplantation of edited normal hCD34+ cells into immunodeficient NOD/SCID IL2?null mice. The most promising approaches based on the evaluation of normal hCD34+ cells will be applied to beta thalassemic hCD34+ cells where the improvement in erythroid parameters both in vitro, and in vivo, will be examined. Our preliminary data on efficient ex-vivo editing of 3 selected genomic loci in normal and 1 in beta thalassemic MPB hCD34+ cells, robust gamma globin reactivation, and durable editing and gamma reactivation in exogeneic recipients are compelling and suggest that this novel approach has the potential to be developed into curative therapies for beta thalassemia.